Leaf-specific chlorophyll a/b binding protein gene promoter from oil palm

ABSTRACT

The present invention discloses a promoter sequence of light-harvesting chlorophyll a/b binding protein pGWLS01 isolated from the oil palm leaf. This promoter enables the manipulation of oil palm leaves for the production of high value-added products via genetic engineering tools. The novel features of the promoter itself which regulate high and specific expression of foreign genes in the leaves will avoid the interference of novel products in the commodity oil extracted from mesocarp and kernel tissues. Furthermore, the promoter is also potentially useful in the production of insect-resistant palm.

TECHNICAL FIELD OF THE INVENTION

The invention is related to type I chlorophyll a/b binding protein genewith abundant and specific expression in the leaf of the Palmae family,its regulatory sequence, and the use of its regulatory sequence forcontrolling the expression of foreign genes to produce high value-addedproducts in the leaves of transgenic plants.

SUMMARY OF THE INVENTION

The present invention relates to the promoter sequence of thelight-harvesting chlorophyll a/b binding protein gene pGWLS01 which wasisolated from the oil palm genome. The presence of this promotersequence will enable the manipulation of oil palm leaves for producinghigh value-added product via the introduction of foreign genes into theoil palm genome using genetic engineering tools. Furthermore, thepromoter is also potentially useful in the production of insectresistant palm for the purpose of crop protection. The novel features ofthe promoter itself which regulate high and specific expression offoreign genes in the leaves will avoid the interference of novelproducts in the commodity oil extracted from mesocarp and kerneltissues. The presence of leaves throughout the plant life cycle willalso enable early harvesting and continuous supply of novel metabolites.

Three different approaches (RT-PCR, cDNA library screening and 5′-RACE)were employed in the isolation of cDNA that encodes for thelight-harvesting chlorophyll a/b binding protein gene (LS01). RT-PCR andscreening of leaf cDNA library resulted in the isolation of partial LS01cDNA sequence with poly(A)⁺ tail. Subsequently 5′-RACE reaction produceda full-length sequence of LS01. This clone was found to exhibit 86% andabove homology at the amino acid level with the deduced amino acidsequences of Lhcb1 of photosystem 11 cDNAs isolated from 9 differentmonocot and dicot plants (GenBank database). Furthermore, the ORF ofLS01 gene also encodes for both transit peptide and mature protein. Thetransit peptide is required for the transportation of LS01 gene into thechloroplast.

The gene copy number of LS01 was determined by Southern blot analysis.The 3′ end gene-specific probe used in the analysis was able todistinguish LS01 from other members of this gene family. Only one copyof this gene is found in the oil palm genome. In the Northern analysis,expression of LS01 transcript was high and strong in the young andmature green leaves. As for yellowish spear leaves, lower level ofexpression was observed. However, the expression of LS01 transcript wasnot detected in the non-photosynthetic tissues such as kernel, mesocarp,germinated seedlings and flower.

The genome walking approach was successfully used for isolating the LS01promoter. The presence of gene-specific primers in both primary andsecondary PCR was able to amplify the genomic clone of interest from apool of digested and adaptor ligated genomic DNA. In addition, the sameapproach was also utilized to study the structure of LS01 genomic clone.It was observed that introns were absent from the LS01 genomic sequence.

The strength and specificity of LS01 promoter was confirmed by atransient assay system and transgenic analysis using model plant,Arabidopsis thaliana. In the transient GUS assay, LS01 promoter wascloned into pB122I vector carrying GUS as a reporter gene after removalof 35S CaMV promoter. As for transient GFP assay, LS01 promoter wascloned into pEGFP promoterless vector carrying GFP as a reporter gene.Both of the plasmids DNA were used in the bombardment of oil palm leaftissues and mesocarp slices as control. Results obtained from the GUSassay and GFP detection confirmed that LS01 promoter was able to drivethe expression of the reporter genes only in the leaf tissue. In theArabidopsis work, the model plant was transformed with Agrobacteriumcarrying a binary vector harboring the leaf-specific promotercontrolling a reporter gene (GUS). Transgenic plant carrying the leafspecific promoter was planted until the third generation in order toobtain a stable integration of the transgenes in the Arabidopsis genome.Results from the GUS staining of the Arabidopsis seedling furtherconfirmed the leaf specificity of LS01 promoter.

Accordingly, it is the primary object of the present invention toprovide a promoter sequence of chlorophyll a/b binding protein geneisolated from oil palm, wherein the promoter sequence exhibitsleaf-specificity.

It is another object of the present invention to provide a promotersequence for controlling leaf-specific expression of foreign genesencoding protein.

It is another object of the present invention to use the complete orpartial sequence of LS01 cDNA or promoter for isolation of promoter orregulatory sequence.

It is yet another object of the present invention to provide arecombinant DNA construct containing LS01 promoter for transformingplant cells, plant tissues or parts of plants.

It is yet another object of the present invention to provide transgenicplants resulting from recombinant DNA constructs, to produce highvalue-added products, monoclonal antibodies, vaccine and other usefulindustrial or pharmaceutical products.

These and other objects of the present invention are accomplished byproviding,

An isolated nucleic acid comprising a regulatory nucleic acid sequencethat is at least 50% identical to the sequence set forth in SEQ ID NO: 1or a complement thereof after optimal alignment.

and

An isolated nucleic acid encoding an amino acid sequence set forth inSEQ ID NO: 2 or an amino acid sequence that has greater than 70%similarity to SEQ ID NO: 2 after optimal alignment.

and

A nucleic acid construct, comprising a nucleic acid as claimed in thepresent invention, wherein the nucleic acid is operably linked to arecombined nucleic acid.

and

A vector comprising the nucleic acid construct as claimed in the presentinvention.

and

The nucleic acid construct as claimed in this invention, wherein therecombined (nucleic acid) encodes a protein that impart insectresistance, production of bioplastic, production of nutraceuticalproducts, production of pharmaceutical macromolecules includingtherapeutic and diagnostic protein, antibodies and vaccines or result inan increase in photosynthetic rate of plant, or result in changes ofplant shade.

and

A cell comprising the nucleic acid construct as claimed in the presentinvention.

and

A transgenic plant comprising the nucleic acid construct as claimed inthe present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the products of RT-PCR using primer CAB(F) and CAB(R). LaneM is the DNA Ladder Mix Marker. Lane 1 and 2 are the 500 bp productsamplified from the pool of expressed gene in oil palm leaves.

FIG. 2 provides the nucleotide (SEQ ID NO: 39) and deduced amino acid(SEQ ID NO:4) sequences of pRTLS01. The amino acids are shown in singleletter codes. The sequence is part of Lhcb gene coding region.

FIG. 3 shows the results of digested phagemids on 1.0% agarose gel. Thephagemids were obtained from in vivo excision of putative clones fromsecondary library screening. Lane M is the DNA Ladder Mix Marker. Lanes1, 3, 5 and 7 are the undigested phagemids. Lanes 2, 4, 6 and 8 are thephagemids digested with EcoR I and Xho 1.

FIG. 4 provides the nucleotide (SEQ ID NO:5) and deduced amino acid (SEQID NO:6) sequences of pLS01 which was obtained from the screening of oilpalm leaf cDNA library. The derived amino acids are presented in singleletter codes. The consensus sequences for polyadenylation signal areunderlined. The stop codon (TGA) is denoted by asterisk (*).

FIG. 5 shows the alignment between deduced amino acid sequences ofpRTLS01 (SEQ ID NO:4) and pLS01 (SEQ ID NO:6). Identical amino acids inboth sequences are presented by asterisk (*). A total of 96% homologywas observed.

FIG. 6 shows the products of SMART RACE that was amplified from the 5′-RACE-Ready cDNA using gene-specific primer, LS 10. Lane M is the DNALadder Mix Marker. Lanes 1, 2 and 3 are the 1.0 kb products of SMARTRACE.

FIG. 7 provides the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQID NO:2) sequences of LS01 complete cDNA sequence. The derived aminoacids are presented in single letter codes. The putative transcriptionstart site is bold and italic. The translation start site is indicatedin boldface. The transit peptide is underlined. The position marked bybrackets denotes the first predicted amino acid of the mature protein.The consensus sequences for polyadenylation signals are underlined anditalic. The stop codon (TGA) is denoted by asterisk (*).

FIG. 8 provides the comparison of the deduced amino acid sequence ofLS01 (SEQ ID NO:2) with Lhcb I amino acid sequences from other plants.The conserved first predicted amino acid of the mature protein wasunderlined. Dots have been introduced to optimize alignment. Asterisk(*) represent identical amino acids. The GenBank accession number of thesequences are as follows: duckweed (L. gibba AAA33396; SEQ ID NO:29),cotton (G. hirsutum AAA18529, SEQ ID NO:30), potato (S. tuberosumAAA80589, SEQ ID NO:31), tobacco (N. sylvestris BAA25388, SEQ ID NO:32),tomato (L. esculentum AAA34137, P07370, SEQ ID NO:33), soya bean (G. maxAAA50172, SEQ ID NO:34), wheat (T. aestivum P04784, SEQ ID NO:35), rice(O. sativa P12330, SEQ ID NO:36) and maize (Z. mays P06671, SEQ IDNO:37).

FIG. 9 provides the results of Northern blot analysis using twodifferent PCR produced probes, LS1 and LS2 containing the entiresequence of LS01 (9a) and 3′- UTR of LS01 (9b), respectively. Total RNA(5 μg/lane) was size fractionated on 1.2% agarose gel and transferred tonylon membrane prior to hybridization with ³²P-labeled LS1 and LS2probes. M, S, Y, F and GS represent total RNA isolated from matureleaves, spear leaves, young leaves, flower and germinated seedlings. Thealphabet ‘w’ represents week after anthesis.

An ethidium bromide stained gel (9c) was included to show the equalloading of total RNA from various oil palm tissues.

FIG. 10 gives the results of Southern analysis for determination of LS01gene copy number in the oil palm genome. A total of 10 μg genomic DNAfrom oil palm leaves was digested with Hind III (Lane H) and Xba I (LaneX) prior to size fractionation on 1.0% agarose gel (10a). The digestedDNA was transferred to nylon membrane and hybridized with ³²P-labeledprobes prepared using the entire sequence of LS01 (10b) and 3′-UTR ofLS01 (10c).

FIG. 11 a shows the amplified PCR products obtained from primary PCR ofGenomeWalker libraries using primers LS14 and AP1. Lane M is the DNALadder Mix Marker. Lanes 1, 2, and 3 are the products amplified from DraI, EcoR V, and Pvu II GenomeWalker libraries, respectively.

FIG. 11 b shows the amplified PCR products obtained from secondary PCRof Dra I GenomeWalker library using primers LS12 and AP2. Lane M is theDNA Ladder Mix Marker. Lanes 1, 2, 3, 4, 5 and 6 are the productsamplified from Dra I GenomeWalker library.

FIG. 12 provides the nucleotide sequence of the oil palm LS01 promoter,pGWLS01 (SEQ ID NO:1). Putative transcription start site is shown initalics. Asterisk (*) represent the overlapping sequences with 5′-UTR ofLS01 gene. Several putative cis-acting elements were identified andunderlined. These consist of initiator element (Inr), I-box, GATA box,CCAAT box, G-box, wound responsive element (WUN), abscisic acidresponsive element (ABA) and heat-shock responsive element (HSE).

FIG. 13 a shows the amplified primary PCR products from Dra 1, EcoR V,Pvu 11 and Stu I GenomeWalker libraries, respectively using primers LS17and API. Lane M is the DNA Ladder Mix Marker. Lanes 1, 2, 3 and 4 arethe products amplified from Dra 1, EcoR V, Pvu 11 and Stu I GenomeWalkerlibraries, respectively.

FIG. 13 b shows the amplified secondary PCR products from Pvu 11GenomeWalker library using primers LS 18 and AP2. Lane M is the DNALadder Mix Marker. Lanes 1 and 2 are the products amplified from Pvu 11GenomeWalker library.

FIG. 14 provides the alignment result of genomic clone, pGWLS1718 (SEQID NO:7) and cDNA clone, LS01 (SEQ ID NO:3). Asterisk (*) represent theconserved nucleotides in both sequences. The translation start site(ATG) is bold and italic. Transcription start site is bold andunderlined.

FIG. 15 shows the results for restriction enzymes analysis of plasmidpLS01 GUS and pLS01GFP using Sma I and Hind 111. Lane M is the DNALadder Mix Marker. Lanes 1 and 3 are the undigested plasmid of pLS01 GUSand pLS01GFP, respectively. Lanes 2 and 4 are the digested plasmid ofpLS01 GUS and pLS01GFP, respectively.

FIG. 16 shows histochemical localization of GUS expression in oil palmleaves bombarded with pLS01 GUS plasmid (16a and 16b) and withoutplasmid (16c). The presence of blue spots were observed in the leavesbombarded with pLS01GUS (16a and 16b) but not in the negative control(16c).

FIG. 17 provides the transient GFP expression in leaf and mesocarptissues bombarded with different promoter-reporter gene constructs. Leafdiscs and mesocarp slices were bombarded with three different plasmids:promoterless pEGFP (negative control), pEGFP driven by LS01 promoter(designated pLS01GFP) and pEGFP driven by constitutive 35S cauliflowermosaic virus promoter (p35SGFP). Expression of GFP was observed in theleaf tissues bombarded with pLS01GFP and p35SGFP. However expression ofGFP was not detected in the mesocarp slices bombarded with pLS01GFP.

FIG. 18 shows restriction enzyme analysis of pB1I01LS01 plasmid usingSma I and Hind 111. Lane M I and M2 is DNA ladder mix and λ Hind IIImarker, respectively. Lanes I and 3 are undigested pBI101LS01 plasmid.Lanes 2 and 4 are pBI101LS01 plasmid digested with Sma I and Hind 111.

FIG. 19 provides PCR analysis of pBI101LS01 plasmid which was isolatedfrom Agrobacterium tumefaciens C58 using LS01 promoter specific primers,LS221c and LS221d. Lane M is the DNA ladder mix marker. Lane I is theundigested pBI101LS01 plasmid which was isolated from Agrobacteriumselected on LB agar plate containing 50 μg/ml kanamycin and rifampycin.Lanes 2, 3 and 4 are the PCR products amplified from plasmid in lane 1.Lane 5 is the undigested pBI101LS01 plasmid which was isolated fromAgrobacterium selected on LB agar plate containing 50 μg/ml kanamycin.Lanes 6, 7 and 8 are the PCR products amplified from plasmid in lane 5.Lane 9 is the water negative control.

FIG. 20 shows PCR analysis of pBI101LS01 plasmid which was isolated fromAgrobacterium tumefaciens C58 using promoter specific primer, LS221c andGUS gene specific primer, GUS-lower. Lane M is the DNA ladder mixmarker. Lane 1 is the amplified PCR product for pBI101LS01 plasmid whichwas obtained from Agrobacterium selected on LB agar plate containing 50μg/ml kanamycin and rifampycin. Lane 2 is the water negative control.

FIG. 21 shows the germination of seeds on kanamycin selection medium toidentify successfully transformed Arabidopsis thaliana progeny.Transformant is resistant towards kanamycin and will grow into green andhealthy seedlings (21 a). However, non-transformant will remain as twoyellowish leaves seedlings even though it was maintained on theselection medium for 1 month (21 b).

FIG. 22 shows the PCR amplification of partial GUS gene to confirm thepresence of pBI101LS01 construct in the putative Arabidopsis thalianatransformant. Genomic DNA extracted from the leaves of transformant wasamplified with primers specific for GUS gene (GUS forward, GUS3FOR andGUS reverse, GUS2REV). Lane M is the DNA ladder mix marker. A fragmentof 348 bp was successfully amplified in Lane 1 and indirectly confirmedthe presence of leaf-specific promoter in the transformant. GUS gene wasnot detected in wild type genomic DNA (Lane 2) and Lane 3 is the waternegative control.

FIG. 23 provides the histochemical GUS staining of 22 days Arabidopsisseedlings from plants transformed with gene construct containing oilpalm leaf-specific promoter and 35S CaMV constitutive promoter. Wildtype plant was used as negative control. In the seedlings transformedwith leaf-specific promoter, blue staining was observed only in the leaf(23a). Blue staining was detected in all the tissues (leaf, stem androot) for plant transformed with constitutive promoter (23b). Bluestaining was not detected in the wild type plant (23c).

BRIEF DESCRIPTION OF THE TABLES

Sequence Sequence Description ID No. identity of sequence 1 pGWLS01 Oilpalm chlorophyll a/b binding protein (LS01) promoter sequence 2 LS01 Oilpalm chlorophyll a/b binding protein amino acid sequence 3 LS01 Oil palmchlorophyll a/b binding protein gene complete ORF sequence 4 pRTLS01Partial LS01 isolated from RT-PCR, amino acid sequence 5 pLS01 PartialLS01 isolated from cDNA library, nucleotide sequence 6 pLS01 PartialLS01 isolated from cDNA library, amino acid sequence 7 pGWLS1718 Genomicsequence obtained from PCR using primers LS17 and LS18 8 CAB(F) Forwarddegenerate primer for amplification of partial LS01 using RT-PCR 9CAB(R) Reverse degenerate primer for amplification of partial LS01 usingRT-PCR 10 LS1 Entire sequence of LS01 cDNA fragment amplified forNorthern blot analysis 11 LS2 3′-UTR of LS01 cDNA fragment amplified forNorthern blot analysis 12 LS10 Antisense primer for amplification of LS1and LS2 13 LS11 Sense primer for amplification of LS2 14 LS12 Nestedgene-specific primer for amplification of pGWLS01 15 LS14 Gene-specificprimer for amplification of pGWLS01 16 LS15 Sense primer foramplification of LS1 17 LS17 Gene-specific primer for amplification ofpGWLS1718 18 LS18 Nested gene-specific primer for amplification ofpGWLS1718 19 AP1 Adaptor-specific primer for amplification of pGWLS01and pGWLS1718 20 AP2 Nested adaptor-specific primer for amplifi- cationof pGWLS01 and pGWLS1718 21 LS221c Sense primer for amplification ofleaf- specific promoter fragment from plasmid pGWLS01 22 LS221dAntisense primer for amplification of leaf- specific promoter fragmentfrom plasmid pGWLS01 23 EGFPN Primer for sequencing of plasmid pLS01GFP24 M13F Forward primer for sequencing of plasmid pLS01GUS 25 M13RReverse primer for sequencing of plasmid pLS01GUS 26 GUS-lower Primerfor detection of gus gene in DNA construct pBI101LS01 27 GUSFOR Forwardprimer for detection of gus gene in genomic DNA of transgenic plant 28GUSREV Reverse primer for detection of gus gene in genomic DNA oftransgenic plant 29 Solanum Solanum tuberosum Lhcb I amino acidtuberosum sequence 30 Nicotiana Nicotiana sylvestris Lhcb I amino acidsylvestris sequence 31 Lycopersicon Lycopersicon esculentum Lhcb I aminoesculentum acid sequence 32 Gossypium Gossypium hirsutum Lhcb I aminoacid hirsutum sequence 33 Glycine max Glycine max Lhcb I amino acidsequence 34 Lemna gibba Lemna gibba Lhcb I amino acid sequence 35 Zeamays Zea mays Lhcb I amino acid sequence 36 Oryza Oryza sativa Lhcb Iamino acid sequence sativa 37 Triticum Triticum aestivum Lhcb I aminoacid aestivum sequence 38 WUN Wound-responsive element 39 pRTLS01Partial LS01 isolated from RT-PCR, nucleotide sequence

DETAILED DESCRIPTION OF THE INVENTION

Sense degenerate primer, CAB(F) and antisense degenerate primer, CAB(R)which were derived from the conserved region of light harvestingchlorophyll a/b binding protein (Lhcb) gene from three differentmonocotyledonous plants consist of maize, rice and grain were used toamplify the related gene from oil palm cDNA pool. Incorporation of theseprimers in the PCR reaction resulted in the isolation of 500 bp Lhcbgene from the single stranded leaf cDNA pool derived from reversetranscription of leaf MRNA (FIG. 1). The nucleotide and deduced aminoacid sequence of this clone was designated pRTLS01 (FIG. 2). BLASTXanalysis using GenBank non-redundant database showed that pRTLS01 has85% and 84% homology with deduced amino acid sequences from maize andrice, respectively. These homologies were expected as the degenerateprimers were designed based on the maize and rice sequences. Sequence ofpRTLS01 was found to code for open reading frame (ORF) of Lhcb gene.

Primary screening of leaf cDNA library using radioactive labeled pRTLS01resulted in the detection of 61 putative oil palm Lhcb clones. Aftersecondary screening, the number of putative clone was reduced to 30. Invivo excision and restriction enzyme analysis of the isolatedrecombinant phagemids prior to sequencing confirmed that all the cloneshave the same insert size of 350 bp (FIG. 3). Based on the nucleotideand deduced amino acid sequences, this clone designated pLS01 consistsof 224 bp ORF, 89 bp 3′-untranslated region (3′-UTR) and poly(A)⁺ tail(FIG. 4). A stop codon(TGA) was observed at position 225 bp.Furthermore, two imperfect consensus signals for polyadenylation, AATAAAand TGTGTTTT were also found at 14 bp and 48 bp, respectively downstreamfrom the TGA stop codon. Both of the imperfect sequences motif were alsoobserved in the 3′-UTR of Scots pine Lhcb cDNA (Jansson & Gustafsson1990). The ORF of pLS01 also showed a very high homology, 96% with partof the ORF from pRTLS01 (FIG. 5).

5′-RACE approach was carried out to obtain the full-length cDNA sequenceand to determine the transcription start site of oil palm Lhcb gene.Based on the sequence information of pLS01 an antisense gene-specificprimer, LS 10 located prior to the poly(A)⁺ tail of this clone wasdesigned. The presence of primer LS10 and adaptor primer from the kit inthe PCR reaction of 5′-RACE-Ready cDNA had resulted in the amplificationof 1.0 kb distinct band (FIG. 6). The nucleotide and deduced amino acidsequences of this clone were designated LS01 (FIG. 7). Analysis on bothof the sequences revealed that LS01 represent the full-length clone ofLhcb gene in oil palm. The adenine at the 5′ end of the LS01 sequence ispredicted to be the putative transcription start site of this gene. Thisclone contains a 5′-untranslated region of 78 bp, an ORF of 795 bp and a3′-untranslated region of 89 bp. The ORF of this clone was found toencode for 265 amino acid protein. A total of 33 amino acids make up thetransit peptide and another 232 make up the mature protein. As Lhcb geneis a nuclear encoded gene, transit peptide is required for thetransportation of this gene into the chloroplast (Mullet 1993). Transitpeptide was also found in the Lhcb gene isolated from other plantspecies such as maize, tobacco, rice and tomato (Demmin et al. 1989).

Table 1 shows the results obtained from BLASTX identity search for LS01clone using

GenBank database. This clone exhibited 86% and above homology with thededuced amino acid sequence of photosystem 11 isolated from 9 differentmonocot and dicot plants. Alignment of LS01 deduced amino acid sequencewith the sequences from other plant species in FIG. 8 showed that theregion coding for mature protein in highly conserved as compared to theregion of transit peptide. Furthermore, a conserved amino acid motif forthe start of mature protein, MRK was also observed in all the plants.

TABLE 1 GenBank Score Percentage of Organisms Identification No. ValueIdentity (%) Solanum tuberosum AAA80589 509 92 Nicotiana sylvestrisBAA25388 508 93 Lycopersicon esculentum AAA34147 508 92 Gossypiumhirsutum AAA18529 506 93 Glycine max AAA50172 499 92 Lemna gibbaAAA33396 496 90 Zea mays P06671 481 88 Oryza sativa P12331 475 89Triticum aestivum P04784 469 86

In the Lhcb multigene family of photosystem II, different types of Lhcb(Lhcb1, Lhcb2 and Lhcb3) can be identified based on the amino acidcharacteristic of transit peptide and mature protein of cDNA clone orthe presence of intron in the genomic clone (Demmin et al. 1989; Chinnet al. 1995). The oil palm full-length LS01 has the comparable structureand number of amino acid as observed in the Lhcb1 of Glycine max(Stockinger & Walling 1994), Gossypium hirsutum (Anderson et al. 1993)and Solanum tuberosum (Fernandez et al. 1995). According to Buetow etal. (1988), Lhcb1 ORF normally comprises of 31 to 37 amino acids fortransit peptide and 231 to 235 amino acids for mature protein. Based onthe results of BLASTX identity search and characteristic of amino acid,it is confirmed that oil palm LS01 belongs to the Lhcb1 gene family.

Northern blot analysis was carried out to determine the expressionpatterns of the oil palm Lhcb gene in various oil palm tissues. Two PCRproduced probes LS1 and LS2 containing entire sequence of LS01 and3′-UTR of LS01 respectively were hybridized with the Northern blots. Itwas observed that both of the probes hybridized to a single transcriptof approximately 1.0 kb (FIGS. 9 a and 9 b). Furthermore, the expressionof LS01 gene was very strong, specific and developmentally regulated inthe leaf tissues. High expression of LS01 was detected in the young andmature green leaves. As for yellowish spear leaves, lower level ofexpression was observed. The expression of LS01 was not detected in thenon-photo synthetic tissues such as kernel, mesocarp, germinatedseedlings and flower.

Intensity of the signal observed in the nylon membrane hybridized withprobe LS 1 is higher than with probe LS2. The differences indicated thatthe entire sequence of LS01 hybridized with the entire mRNA transcriptthat contained the conserved coding region of Lhcb gene. Whereas the3′-UTR probe enable the specific analysis of individual gene becauseonly LS01 transcript was hybridized. These results revealed that besideLS01, other members of the Lhcb gene could be present in the oil palmleaves.

Genomic Southern analysis was performed to determine the gene copynumber of the oil palm LS01. The same PCR produced probes for Northernanalysis were used to hybridize to the Southern blots containing genomicDNA digested with Hind III (Lane H) and Xba I (Lane X). It was observedin FIG. 10 a that the entire sequence of LS01 hybridized to 4 differentfragments in both lanes. However, in the membrane hybridized with the3′-UTR probe, only one fragment was detected in each lane (FIG. 10 b).It was found that these bands, 10 kb in Lane H and 2.2 kb in Lane Xmigrated in the same distance as one of the fragment in the membranehybridized with the entire sequence of LS01. These results confirmed thepresence of only one copy of LS01 gene in the oil palm genome.

In order to obtain the 5′ upstream regulatory sequence of LS01,genome-walking approach was performed using GenomeWalker kit andAdvantage Genomic PCR kit from CLONTECH. In the primary PCR ofGenomeWalker libraries using a 29-mer gene-specific primer, LS14 andadaptor primer, AP1, genomic fragments with different sizes wereamplified from Dra I, EcoR V and Pvu II digested libraries (FIG. 11 a).The largest fragment of 1.2 kb was amplified from the Dra I digestedlibrary. Whereas PCR products of approximately 800 bp was amplified fromboth EcoR V and Pvu II libraries. Since the Dra I digested librarycontained the largest PCR product, it was selected as a template forsecondary PCR. The library was diluted 50× and 1 μl was used in the PCRreaction with a 30-mer nested gene-specific primer, LS12, and nestedadaptor primer, AP2 (5′ACTATAGGGCACGCGTGGT3′; SEQ ID NO:20). SecondaryPCR using Dra I library resulted in the amplification of 1.0 kb PCRproduct (FIG. 11 b). Since the primer LS12 located 228 bp upstream ofthe primer LS14, smaller size of amplified genomic fragment around 1.0kb was expected. The fragment was subsequently cloned into TOPO-pCR®IIvector and the recombinant clone designated pGWLS01 was sequenced usingM13 forward and reverse primer.

Sequencing result of pGWLS01 was shown in FIG. 12. A total of 932 bpnucleotides upstream of the putative transcription start site coding forthe promoter region. As for the 58 bp nucleotides downstream of theputative transcription start site, this region overlaps with the 5′-UTRof LS01. At the expected distant 32±7 bp upstream to the transcriptionstart site, no TATA-box consensus sequence was identified (Joshi 1987).However, at position −1 to −7 upstream from the transcription startsite, an initiator element (Inr) which is pyrimidine rich, PyTCANTPyPywas observed (Nakamura et al 2002). This finding revealed that the oilpalm LS01 promoter is a TATA-less promoter and initiation of basaltranscription could be directed by the Inr motif. The absence of TATAboxes in the majority of nuclear encoded photosynthesis genes werereported previously by Nakamura et al (2002). Their studies on 232promoter sequences strongly suggest that TATA-independent transcriptionmechanisms play an important role in the regulated expression ofphotosynthesis nuclear genes.

Furthermore, at the distal region of the promoter, a few interestingcis-acting elements were identified. Light-responsive elements such asGATA, CCAAT, G- and I-box were commonly found in the light-responsivepromoter (Arguello-Astorga & Herrera-Estrella 1998). Two separatedregions that contain GATA and CCAAT motifs at position −88 and −65relative to the transcription start site were postulated to bephytochrome-responsiveness. These motifs were also identified in theLhcb promoter of Lemna gibba and other plants (Kehoe et al. 1994). Inthe upstream region of the promoter, a putative wound-responsive element(WUN) CAAATTCCAAA (SEQ ID NO:38) nearly identical to the WUN ofpathogenesis-related gene, AAATTTCCT in potato was identified atposition −464 (Matton et al. 1993). Whereas at position −699 and −878,an abscisic acid-responsive element (Knight et al. 1992) and heat-shockresponsive element (Pastuglia et al. 1997) were identified,respectively. The presence of these elements indicated that theexpression of LS01 gene could be regulated by environmental cues such aslight, mechanical wounding, abscisic acid and heat.

GenomeWalker kit was also used to determine the presence of intron inthe LS01 gene. In the primary PCR with primer LS17, an intense genomicfragment was amplified from EcoR V and Pvu II GenomeWalker libraries. Noband was observed in Dra I library and only a small fragment about 0.3kb was amplified from Stu I library (FIG. 13 a). Since the fragment fromPvu II library is slightly larger than EcoR V, it was selected forsecondary PCR. Further amplification of this template with nestedgene-specific primer, LS 18 resulted in the isolation of a 0.9 kbgenomic clone (FIG. 13 b). Alignment between this sequence, designatedpGWLS1718 with full-length LS01 cDNA sequence was shown in FIG. 14. Itwas found that nucleotides upstream to the transcription start site weresimilar to the proximal region of LS01 promoter. Whereas a total of 508bp nucleotides downstream to the transcription start site were identicalto the coding region of LS01 gene. No introns sequences were presence inthis clone. Such a criterion is only observed in the Lhcb1 gene which istypically lack of introns (Arguello-Astorga & Herrera-Estrella 1998).This result further confirms that oil palm LS01 belongs to the Lhcblgene family.

Transient expression assay was carried out for analyzing the strengthand specificity of oil palm leaf-specific promoter. A 900 bp promoterwhich was amplified from pGWLS01 using primer LS221c and LS221d has beensuccessfully ligated into promoterless pBI221 and pEGFP vector carryingGUS and GFP, respectively as reporter gene. Restriction analysis of therecombinant plasmids designated pLS01GUS and pLS01 GFP using Sma I andHind III was shown in FIG. 15. Digested pLS01GUS showed the presence of5.7 kb pB1221 vector and 900 bp promoter. As for pLS01GFP, fragments forvector and promoter were also observed. The size of the pEGFP vector was4.2 kb. Analysis of the sequencing results confirmed that LS01 promoterwas ligated in the correct orientation in both recombinant plasmids.

In the histochemical localization of GUS expression, GUS enzyme (EC3.2.1.31) which was encoded by the uidA locus will catalyse the cleavageof the substrate 5-bromo-4-chloro-3 indoyl glucuronide (X-Gluc).Precipitation of blue dye at the site of enzyme activity was obtainedthrough oxidative dimerization of the indoxyl derivative. It was foundthat the presence of oxidative catalyst such as ferricyanide andferrocyanide mixture could enhance this dimerization process (Jeffersonet al, 1987). In the studies, 2 days of incubation in GUS stainingsolution resulted in the detection of 60 blue spots in the leaf tissuesbombarded with pLS01GUS at 1350 psi helium pressure and 9 cm distancefrom macrocarrier to target tissues. Moreover, it was also observed inFIG. 16 that some blue spots could be attributed to a single blue cell(16a), but in most of the cases several adjacent cells were also stained(16b). This type of enzyme localization was also detected in the workcarried out by Chowdhury et al, (1977). As for the negative control inFIG. 16c, the presence of blue spot was not observed.

Specificity of LS01 promoter was further proven by the result obtainedfrom GFP detection. Bombardment of leaf discs and control mesocarpslices with pLS01GFP showed a very promising result. Using thisconstruct, GFP expression was detected only in the leaf but not in themesocarp slices (FIG. 17). As for the construct driven by constitutive35S promoter, green fluorescence spots were detected in both tissues. Noexpression of GFP was detected in the tissues bombarded withpromoterless pEGFP.

For a stable integration of LS01 promoter into the Arabidopsis genome,the first step involved the construction of a suitable recombinantbinary vector and cloning in E. coli. Transformation of the ligatedproduct containing the oil palm leaf-specific promoter (LS01) and pBI101binary vector into E.coli strain DH5α resulted in the isolation of twosingle bacteria colonies. The recombinant plasmids designated pBI101LS01were then extracted from these colonies. As shown in FIG. 18, twodifferent sizes of bands which were 12.5 kb vector and 0.9 kb LS01fragment were observed after the pBI101LS01 plasmid was digested withSma I and Hind 111. This analysis confirmed that LS01 has beensuccessfully cloned into pBI101. Whereas analysis of the sequencingresults showed that LS01 was ligated into pB101 in the correctorientation.

In order to enable the transfer of construct of interest intoArabidopsis, pBI101LS01 plasmid was transformed into the Agrobacteriumtumefaciens strain C58 which has often been called ‘nature's geneticengineer’ using electroporation method. After 3 days incubation at 28°C., Agrobacteriumcolonies were observed in the agar plate containingkanamycin. It was found that the actual volt of 2.21 kV and timeconstant of 5.2 mseconds yielded bigger size colonies. Subsequently, atotal of 8 transformants were grown in LB broth containing kanamycin andplasmids were prepared using QIAPREP spin miniprep kit (QIAGEN). On theother hand, glycerol stocks of the same plasmids were also restreaked onLB plate containing rifampycin and kanamycin. Plasmids were alsoprepared from these transformants. As shown in FIG. 19, transformantsobtained from two different antibiotic selection plates showed thepresence of 0.9 kb LS10 promoter fragment. This result suggested thatkanamycin alone was able to select recombinant Agrobacterium. In orderto verify accurately the presence of T-DNA plasmid in the transformants,PCR analysis was repeated using leaf promoter and GUS specific primers.It was observed in FIG. 20 the amplification of 2.7 kb PCR product. Theexpected size comprised of 1.8 kb GUS gene (Mayer et al., 2001) and 0.9kb LS01 promoter fragment. Based on these results, the transformedAgrobacterium can be used for transforming Arabidopsis thaliana.

In planta transformation of Arabidopsis was performed via floral dipmethod. According to Weigel and Glazebrook (2002), this method was ableto give transformant frequency of 0.1 to 1%. In the work carried out byClough and Bent (1998), they concluded that inflorescence developmentalstage and inoculation medium were the most important factors thatdetermine the efficiency of floral dip transformation. Plants with themaximum number of unopened floral buds were the most susceptible stagefor transformation. Whereas with the presence of 5% sucrose in theinoculum, a total of 1.62% transformed Arabidopsis was obtained. As forsurfactant SILWET L-77, this component greatly enhances the entry ofbacteria into relatively inaccessible plant tissues. In this study,plants with many immature floral bud and few opened flowers were chosenfor dipping in the inoculation medium containing LB broth, 5% sucroseand 0.05% SILWET L-77. After 8 weeks on the soil, seeds were collectedfrom the T₁ plant.

Selection of putative transformant from T₁, seeds was carried out on thekanamycin selection medium. After 14 days, transformant was observed inone of the plate. The transformant was a kanamycin resistant seedlingthat produced green leaves and able to develop into a mature Arabidopsisplant (FIG. 21 a). As for non-transformant, the seedling will only havetwo yellowish leaves and the growth was retarded (FIG. 21 b). On thethird week, the number of adult leaves of the transformed Arabidopsishas increased from 3 to 5 leaves. At this stage, the seedling wastransplanted into soil. Seeds collected from this plant were designatedas T₂.

Screening of 16 plants from T₂ generation resulted in the isolation ofone homozygous line (designated as T₁P₁,/T₂P₄) which exhibited 100%survivor rate on the kanamycin selection medium. PCR analysis hadsuccessfully amplified a 348 bp partial GUS gene from the leaf genomicDNA of this homozygous plant (FIG. 22). This further confirmed thepresence of pBI101LS01 construct in the transformed plant. In addition,GUS assay of 22 days seedlings also showed the specific expression inthe leaf tissue. GUS staining was observed only in the leaf tissue butnot in the stem and root (23a). However for the plant transformed withthe gene construct containing 35S constitutive promoter, all the tissuesstated above were stained blue (23b). As for wild type plant, no bluestaining was obtained (23c).

Based on the results from transient expression assay and stabletransformation in the Arabidopsis, it can be concluded that the LS01promoter was able to drive the expression of transgenes specific to theleaf tissue. Furthermore, successful transformation of this promoterinto the Arabidopsis also showed that it can be used in the heterologousplant system.

EXAMPLES Example 1 Amplification of Lhcb Gene Via Reverse Transcriptionand Polymerase Chain Reaction (RT-PCR) Approach

First strand cDNA was synthesized from leaf mRNA in 25 μl reactioncontaining 5 μg leafmRNA, 4 μl 5× first strand buffer, 2 μl 0.1 M DTT, 5μl 2 mM dNTP and 1 μl SUPERSCRIPT II reverse transcriptase (200 U/μl )(Gibco BRL Life Technology Inc. N.Y., USA) at 42° C. for 1 hour. Thesolution was phenol-chloroform extracted, ethanol precipitated and thepellet was dissolved in 25 μl sterile water. The cDNA was subjected toRNA hydrolysis using 12.5 μl 0.15 N NaOH and 1 μl 0.5 M EDTA at 68° C.for 15 min. This was followed by neutralization of the reaction byaddition of 12.5 μl Tris-HCI, pH 8.0; 12.5 μl 1 N HCl and 17.3 μl 7.5 Mammonium acetate prior to ethanol precipitation. The cDNA pellet wasdissolved in 25 μl sterile water and the concentration of cDNA wasdetermined using ethidium bromide plate.

PCR was carried out with the presence of degenerate primers[CAB(R)-5′CNGGRTCNGCDATRTGRT3′ (SEQ ID NO:9) andCAB(F)-5′GCNGAYCCNGARACNTTY3′ (SEQ ID NO:8)] which were designed basedon the conserved region of known chlorophyll a/b binding protein genefrom maize, grain and rice. The 50 μl PCR mixture contained 1 μl cDNA(50 ng),5 μl 10× buffer, 1 μl 10 mM dATP, 1 μl 10 mM dCTP, 1 μl 10 mMdGTP, 1 μl 10 mM dTTP, 2 μl 15 μM CAB(R), 2 μl 15 μM CAB(F), 5 μl 25 μMMgCl₂, 30.5 μl sterile water and 0 μl Amplitaq DNA polymerase(5 U/μl).The PCR reaction was placed in the Perkin Elmer 9700 thermo cycler withthe following conditions: 94° C., 5 min for 1 cycle; followed by 94° C.,1 min; 43° C., 1 min and 72° C., 1 min 30 sec for 40 cycles and finally72° C., 10 min for 1 cycle. The expected fragment was purified usingQIAQUICK Gel Extraction Kit (QIAGEN), cloned into TOPO-pCR®II vectorfrom TOPO-TA Cloning Kit (Invitrogen) before subjected to automatedsequencing with ABI 377 PRISM. Analysis of the nucleotide sequences wereperformed using DNASIS Max version 1.0 prior to database homologysearch.

Example 2 Screening of Leaf CDNA Library With cDNA Probe Generated FromRT-PCR

A total of 200,000 plaques from leaf cDNA library constructed in Uni-ZAPXR vector (Stratagene) were plated based on Sambrook et al (1989) andplaque lift was performed as described by Siti Nor Akmar et al (1995).The plaques lifted membranes were first treated with denaturation buffer(0.5 N NaOH, 1.0 M NaCl) for 10 min, followed by neutralization buffer(0.5 M Tris-HCl, pH 8.0; 1.5 M NaCl) for 5 min and 2×SSC for 5 min priorto optimal crosslinked with UV light.

Prehybridization of the membranes were carried out at 65° C. in 5×Denhardt's solution (1× Denhardt's solution is 0.02% each Ficoll 400,bovine serum albumin and polyvinylpyrrolidone), 5× SSPE (1× SSPE is 0.18M NaCl, 10 M NaH₂PO₄, pH 7.5, 1 mM EDTA), 0.5% SDS and 100 μg/mldenatured herring sperm DNA. Hybridization of the membranes wasperformed using the same hybridization buffer with the presence of³²P-labelled pRTLS01 at 65° C. The probe was labeled with ³²P-dCTP usingMEGAPRIME DNA Labeling Kit from Amersham Pharmacia Biotech. Afterovernight hybridization, the membranes were washed at 65° C. with 4×SSPE, 0.1% SDS; followed by 2× SSPE, 0.1% SDS and 0.5× SSPE, 0.1% SDS.These membranes were then exposed to x-ray film for 24 hours at −80° C.

Based on the signal detected on x-ray film, putative plaques were coredout and placed into SM buffer (100 mM NaCl, 10 mM MgSO₄.7H₂0, 0.05 MTris-HCI and 0.01% gelatin) containing 0.3% chloroform. The phage lysatewas subjected to PCR analysis with the presence of primer T7 andgene-specific primers. The PCR conditions were as follows: 95° C., 5 minand 80° C., 45 min for 1 cycle; followed by 95° C., 1 min; 60° C., 1 minand 72° C., 1 min 30 see for 30 cycles and 1 cycle of final extension at72° C. for 10 min. Amplified products were visualized on 1.2% agarosegel.

Recombinant phage was in vivo excised according to the manufacturer'sinstruction (Stratagene). The purified pBluescript phagemid was digestedwith EcoR I and Xho I to confirm the length of cDNA insert.

Example 3 Rapid Amplification of 5′-cDNA Ends (5′-RA CE)

A full-length sequence of chlorophyll a/b binding protein gene wasisolated via 5′ RACE using SMART RACE cDNA amplification Kit andAdvantage 2 PCR Kit from CLONTECH, Laboratories, Inc., USA. The reactionfor first strand cDNA was initiated by incubation of 1 μg total RNA fromleaf with 1 μl 5′-CDS primer, 1 μl SMART II A oligo and 2 μl sterilewater at 70° C. for 2 min. The reaction was immediately cooled on iceprior to addition of 2 μl 5× first strand buffer, 1 μl 20 mM DTT, 1 μl10 mM dNTP mix and 1 μl reverse transcription PowerScript. This wasfollowed by incubation at 42° C. for 1.5 hours. The reaction was stoppedby addition of 200 μl Tricine-EDTA buffer and incubation at 72° C. for 7min.

A total of 2.5 μl single stranded 5′-RACE Ready cDNA was added to thePCR mixture containing 34.5 μl PCR grade water, 5 μl 50× Advantage 2polymerase buffer, 5 μl 10× UPM primer and 1 μl 10 μM gene-specificprimer, LS10 (5′TAATGCACACCACGCCAACAATTTCAATTC3′; SEQ ID NO:12) whichwas designed based on the sequence of pRTLS01. The PCR reaction wascarried out using Perkin Elmer9700 thermo cycler with the followingconditions: 94° C., 5 sec; 68° C., 10 sec and 72° C., 3 min for 25cycles. The expected fragment was purified, cloned into TOPO-pCR®IIvector from TOPO-TA Cloning Kit (Invitrogen) before subjected toautomated sequencing with ABI 377 PRISM. Analysis of the nucleotidesequences were performed using DNASIS Max version 1.0.

Example 4 Northern Blot Analysis

Total RNA was extracted from various tissues of oil palm according tothe method of Rochester et al. (1986).

Two different fragments containing complete nucleotide sequence and 3′untranslated region (3′-UTR) of chlorophyll a/b binding protein genewere used as probes in the Northern and Southern analysis. The completenucleotide sequence of LS01 was generated through amplification ofplasmid LS 10.3 with primer LS 10 (5′TAATGCACACCACGCCAACAATTTCAATTC3′;SEQ ID NO:12) and LS15 (5′GCACCTACCCAACAGCATTTCCATTGG3′; SEQ ID NO:16).Whereas 3′-UTR region was amplified using LS10 and LS11(5′GCCTGGCAACCTTAATTAATTTGGTGCTTAG3′; SEQ ID NO:13) primer pair. TheExpected fragments were purified using QIAQUICK Gel Extraction Kit(QIAGEN) and labeled with ³²P-dCTP using MEGAPRIME DNA Labeling Kit fromAmersham Pharmacia Biotech.

Northern blot analysis has been carried out according to the method ofMcMaster & Carmichael (1977) and Kroczek & Siebert (1990). In thisstudy, 5 μg of total RNA was heat denatured at 55° C. for 15 min in 18μl GFP mixture containing 78% (v/v) deionized formamide, 16% deionizedglyoxal and 10 mM sodium phosphate buffer. After heat denaturation, theRNA was cooled immediately on ice prior to electrophoresis on 1.2%agarose gel with 40 mM× TAE, pH 7.2 as electrophoresis buffer. The RNAwas transferred to HYBOND-N⁺ membrane (Amersham Pharmacia Biotech) via avacuum blotter (60 psi, 6 hours) with the presence of 20×SSC as transferbuffer.

Prehybridization of the membrane was performed at 65° C. for 4 hours in5×SSC (1×SSC is 0.15 M NaCl, 15 mM trisodium citrate), 5× Denhardt's (1×Denhardt's is and 0.02% each Ficoll 400, bovine serum albumin andpolyvinylpyrrolidone), 0.5% SDS 100 μg/ml denatured herring sperm DNA.This was followed by overnight hybridization of the membrane with³²P-labeled probe at 65° C. Washing of the membrane was performed with4×SSC/0.1% SDS at 65° C. for 15 min, followed by 2×SSC/0.1% SDS at 65°C. for 15 min. Exposure to x-ray film was carried out at −80° C. for 48hours.

Example 5 Southern Blot Analysis

Genomic DNA was extracted from oil palm spear leaves according to themethod of Doyle & Doyle (1990).

A total of 20 μg genomic DNA was digested with Hind III and Xba I. Thedigested products were size fractionated on 1.0% agarose gel at 100 vfor 5 hours in 1× TAE, pH 7.9. This was followed by immobilization ofthe DNA onto nylon membrane via vacuum blotting of the gel at 60 psi for1 hour with the presence of 0.4 N NaOH as transfer buffer. At the end ofthe process, the membrane was rinsed with 2×SSC prior toUV-crosslinking. Hybridization and washing of the blot was performed asstated above for Northern blot analysis.

Example 6 Promoter Isolation

Leaf-specific promoter was isolated following the standard protocolstated in the manual of Universal GenomeWalker Kit and Advantage GenomicPCR Kit from CLONTECH Laboratories, Inc. Two antisense gene-specificprimers, designated LS14 (5′GTGTCCCACCCATAGTCACCGGGGAATTC3′; SEQ IDNO:15) and LS12 (5′GATGATGCCTTGGAGATGGGAGCGGTGATC3′; SEQ ID NO:14) weredesigned based on the 5′-terminal of the coding region of LS01 andwithin 5′-UTR of LS01, respectively. A total of four GenomeWalkerlibraries were obtained through digestion of 2.5 μg leaf genomic DNAwith Dra 1, EcoR V, Pvu 11 and Stu I prior to ligation with theGenomeWalker adaptor. An aliquot of 12 μl of these libraries were usedin the adaptor primer, AP2 (5′ACTATAGGGCACGCGTGGT3′; SEQ ID NO:20),supplied in the kit. PCR conditions were carried out as primary PCRreaction with the presence of antisense gene-specific primer, LS 12, andadaptor primer, AP1 (5′GTAATACGACTCACTATAGGGC3′; SEQ ID NO:19) providedwith the kit. This was followed by secondary PCR of 50× diluted primarylibrary with antisense nested gene specific primer, LS 14 andrecommended in the GenomeWalker Kit manual using Perkin Elmer 9700thermo cycler. The expected band was purified from agarose gel usingQIAquick

Gel Extraction Kit (QIAgen), cloned into TOPO-pCR®II vector from TOPO-TACloning Kit (Invitrogen) prior to sequencing using M13 reverse andforward primers.

Genome walking approach was also used to study the structure of LS01gene. Primary PCR of the GenomeWalker libraries was carried out with thepresence of antisense gene-specific primer, LS17(5′CGAAGTTGGTGGCGTAGGCCCAAGCATTG3′; SEQ ID NO:17) from 3′-terminal ofthe coding region of pLS01 and primer AP 1. Followed by secondary PCRwith antisense nested gene specific primer, LS 18(5′CTCTGAGCATGGATCAAGCTCGGGTTGCC3′; SEQ ID NO:18) from 5′-terminal ofthe coding region of pLS01 and primer AP2. The expected band waspurified, cloned and sequenced as above.

Example 7 Cloning of the Leaf-specific Promoter into pBI221 and pEGFPVector

Leaf specific promoter (922 bp) was amplified from plasmid pGWLS01 usingsense and antisense primer. The sense primer, LS221c(5′CCCAAGCTTCCATATCTGGCTCG3′; SEQ ID NO:21) was introduced with Hind IIIsite at the 5′ end. As for antisense primer, LS221d(5′TCCCCCGGGCAATGGAAATGCTG3′; SEQ ID NO:22), 5′ end was introduced withSma I site. These primers, 2 μl of 15 μM stock, were used to amplify thepromoter in 50 μl PCR reaction contained 4 μl dNTP (10 mM each), 250 ngplasmid pGWLS01, 5 μl 10× buffer, 5 μl 25 mM MgCl₂ and 2.5 U AMPLITAQDNA Polymerase from Perkin Elmer. PCR conditions were as follows: 95°C., 5 min for 1 cycle; followed by 95° C., 1 min; 56° C., 1 min and 72°C., 1 min 30 sec for 30 cycles and 1 cycle of final extension at 72° C.for 10 min.The PCR product was purified from primers, nucleotides,polymerases and salts using QIAQUICK PCR Purification column fromQIAGEN. Plasmid pB1221 carrying GUS as reporter gene and promoterlessplasmid, pEGFP carrying GFP as reporter gene were prepared using QIAPREPMiniprep kit from QIAGEN. Fragment of LS01 promoter, plasmid pB1221 andpEGFP were digested first with Sma I (Fermentes) at 30° C. for 6 hours.Digestion with second restriction enzyme, Hind III (Fermentes) wasperformed at 37° C. for 16 hours. The digested products were analyzed on1.0% agarose gel and expected fragments were purified from the agarosegel using QIAQUICK Gel Extraction Kit from QIAGEN.

Digested LS01 promoter and promoterless pB1221 at a molar ratio of 4:1were incubated at 50° C. for 5 min. After immediate cooling, the vectorand insert mixture were added into ligation mixture containing 1.5 μl10× ligase buffer and 1.5 μl T4 DNA ligase (1 U/μl) prior to overnightincubation at 16° C. Ten microlitres of the ligation mixture were usedfor transformation with competent cell of E.coli DH5α as described bySiti Nor Akmar Abdullah (1999). Blue/white selection of recombinantclones were carried out on LB plate containing 40 μl of 20 mg/ml5-bromo-4-chloro3-indolyl-β-D-galactopyranoside (X-gal) and 40 μl of 20mg/ml isopropyl β-D-thiogalactopyranoside (IPTG). Plasmid of therecombinant clone, designated pLS01GUS was prepared using QIAPREP SpinMiniprep Kit (QIAGEN) and digestion with restriction enzymes Sma I andHind III was performed to confirm the insert size. Lastly, the plasmidwas sequenced using M13F (5′GTAAAACGACGGCCAG3′; SEQ ID NO:24) and M13R(5′CAGGAAACAGCTATGAC3′; SEQ ID NO:25) primers. Cloning of the digestedLS01 promoter into promoterless pEGFP was also carried out as above andthe recombinant clone, pLS01 GFP was sequenced using EGFPN primer(5′CGTCGCCGTCCAGCTCGACCAG3′; SEQ ID NO:23).

Example 8 Promoter Analysis Via Histochemical GUS Assay and GFPDetection

Preparation of Tissue Slices

Oil palm green leaves were collected from seedling palm in MPOB nursery.The tissues were soaked in RBS for 15 min before subjecting to surfacesterilization using 20% of CLOROX for 15 min in the laminar flow. Theleaves were rinsed twice with sterile water, cut into segments of 1.0cm² and were flattened on Murashige and Skoog medium (Duchefa,Biochemicals Plant Cell and Tissue Culture, Haarlem, Netherlands) withthe lower epidermis facing upward. Leaf discs were kept at 28° C. for 24hours and were illuminated before and after bombardment with chimericgene construct.

Preparation of Gold for Particle Bombardment

A total of 1 ml absolute ethanol was added to 0.06 g of 1.0 micron goldparticles. The mixture was mixed by vortexing at high speed. Aftercentrifugation at 10,000 g for 1 min, the supernatant was removed fromthe gold pellet. These sterilization steps were repeated for 3 times. Atthe final sterilization, 1 ml sterile water was added to the goldpellet. Sonication of the gold pellet was repeated 3 times before thegold was resuspended in 1 ml sterile water. The gold can be stored at−20° C. for up to 6 months.

Precipitation of DNA Onto Gold Microprojectiles

An approximately 10 μg of plasmid pLS01GUS was added to 2 μg of goldparticles in a microcentrufuge tube. While vortexing, 100 μl 2.5 Mcalcium and 40 μl 0.1 M spermidine was added. The mixture was vortexedfor 3 min prior to centrifugation at 10,000 g for 1 min. The supernatantwas removed and the microcarrier was resuspended in 65 μl absoluteethanol. The DNA-coated microcarrier can be kept at −20° C. until used.

Microprojectile Bombardment

Leaf discs were bombarded with gene construct using the BioRad(Hercules, Calif., USA) PDS-1000/Helium-driven Particle Delivery System.Before bombardment, the machine chamber, rupture disc, stopping screen,macrocarrier, macrocarrier holder and red caplugs were sterilized withabsolute ethanol. An aliquot of 8 μl DNA-coated microcarrier was loadedonto the center of the macrocarrier and air dried for 10 minutes. Theleaf discs were bombarded using the following conditions: 1350 psihelium pressure with 9 cm distance from macrocarrier to target tissues.As for mesocarp slices, bombardment was performed at 1550 psi with 9 cmdistance from macrocarrier to target tissues. Leaf discs bombardedwithout plasmid DNA were used as negative control in GUS assay. Whereasin GFP, leaf discs and mesocarp slices bombarded with promoterless pEGFPwere used as negative control. After bombardment, the leaf tissues wereincubated for 24 hours at 28° C. in the light prior to GUS assay and GFPdetection.

Histochemical GUS Assay

GUS activity was measured histochemically following the method describedby Jefferson et al. (1987). The leaf tissues were incubated infilter-sterilized GUS staining buffer (0.1 M sodium phosphate buffer, pH7.0; 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mg/ml5-bromo-4-chloro-3-indoyl glucuronide/X-Gluc, 0.2% Triton X-100 and 10mM EDTA) for up to 2 days at 37° C. in the dark. After staining, thetissues were fixed in fixative mix (10% formaldehyde, 50% ethanol and 5%glacial acetic acid) and the leaves chlorophyll was removed using 80%ethanol. The presence of blue spots were observed using LEICA QF550WSystem.

Detection of Green Fluorescent Protein (GFP)

Microscopic detection of GFP was carried out using LEICA MZ 12.5microscope with blue light. Image of the GFP was captured using ImageManager 50 computer software.

Example 9 Promoter Analysis Using Arabidopsis thialiana (Model PlantSystem)

Preparation of PB1101LS01 Construct

Fragment of leaf-specific promoter from plasmid pGWLS01 and promoterlessbinary vector pB 101 (at a molar ratio of 13:1) which have been digestedwith Sma I and Hind III were heat treated at 55° C. for 5 min. This wasfollowed by immediate cooling and addition of ligation Mixturecontaining 1.5 μl 10× ligase buffer and 1.5 μl T4 DNA ligase (1 U/μl)prior to overnight incubation at 16° C. Fifty microlitres of theligation mixture were then used to transform competent cell E.coli DH5αand selection of recombinant clones were carried out on Luria-Bertani(LB) plate containing 50 μg/ml kanamycin. Plasmid of the recombinantclone, designated pBI101LS01 was prepared using QIAPREP spin miniprepkit (QIAGEN) and digestion with restriction enzymes Sma I and Hind IIIwas performed to confirm the insert size. Lastly, the plasmid wassequenced using M 13F and M13R primers.

Electroporation

A total of 100 ng binary plasmid pBI101LS01 was mixed on ice with 100 μlcompetent cell of Agrobacterium tumefaciens strain C58 in a prechilledGene Pulser Cuvette (0.2 cm electrode gap). The mixture was subjected toelectroporation using Electroporator Gene Pulser®II (Bio-Rad) with thefollowing conditions: capacitance: 1.0 μF, voltage: 2.2 kV and timeconstant: 5 to 10 mseconds. Immediately after electroporation, 1 ml ofLB broth was added to the cuvette prior to incubation at 28° C. for 4hours. The culture was incubated without shaking for the first 2 hoursand continued by gentle shaking at 150 rpm for the following 2 hours.After incubation, the cells were collected by centrifuging briefly at4000 rpm for 1 min. The pellet was resuspended in 40 μl of LB broth andthe cells were plated on LB plate containing 50 μg/ml kanamycin. Theplate was incubated for 3 days at 28° C. and PCR was performed usingprimers specific for leaf promoter and GUS gene (GUS-lower,5′CATTGTTTGCCTCCCTGCTGCGGTT3′; SEQ ID NO:26) to verify the presence ofthe recombinant binary vector, pB101LS01 in the Agrobacterium.

Growing of the Arabidopsis Plant

Seeds from Arabidopsis thaliana ecotype Columbia were germinated andgrown to flowering stage in a flower pots filled with a soil mixture oftwo-thirds Steven Dutch potting mix and one-third vermiculite in agrowth chamber. The growth conditions were 16 hours lights (80-100μmol/m²) at 22° C. 8 h dark at 20° C., with a relative humidity of 75%.In order to obtain more floral bud per plant for dipping, primaryinflorescences were clipped to encourage the emergence of secondarybolts.

In Planta Transformation via Floral Din

A single colony of Agrobacterium tumefaciens strain C58 harboring thebinary plasmid pBI101LS01 was grown overnight at 28° C. with shaking(220 rpm) in LB broth containing 50 μg/ml kanamycin. The overnightcultures were diluted to 1:10 using LB broth and grown for approximately8 hours to obtain OD₆₀₀ of 0.6-0.8. Cells were harvested bycentrifugation at 5000 rpm for 15 min. The pellet which was pink incolor was then resuspended in 5% (w/v) sucrose. The culture wastransferred to a square container and SILWET L-77 was added to 0.05%(v/v). Plants were inverted into this suspension and dipped for 10seconds. Plants were then removed and the flower pots were enclosed withplastic bags. Top of the plastic bags were closed with paper clip for 2days to maintain high humidity. After 48 hours, the paper clip wasremoved and the plants were grown under normal growing conditions. Theplant which was designated as T₁ were grown to maturity and harvestedwhen the siliques were brown and dry.

Screening and Selection of Putative Transformant

Harvesting of Transgenic Seeds

Seven weeks after floral dip, putative transgenic seeds were harvestedby gentle pulling of Dry siliques through fingers over a piece of cleanpaper. The seeds were sieved to remove the pod materials. Clean seedswere stored in the EPPENDORF tube and kept at 22° C. in the dessicator.

Sterilization of Transgenic Seeds

The seeds were suspended in sterile water containing 1% TWEEN 20 (v/v)in an EPPENDORF tube. The seeds were vortexed, centrifuged and rinsedwith sterile water 3 times prior to soaking in 25% Clorox for 20minutes. Clorox was then removed by washing the seeds with sterile water3 times. Lastly the seeds were subjected to surface sterilization with70% ethanol for 1 minute. The seeds were rinsed again with sterile waterbefore it can be used for selection.

Selection for Homozygous Line Carrying Leaf-Specific Promoter

Sterilized seeds were plated on Murashige and Skoog medium supplementedwith 50 μg/ml kanamycin. The selection plates were cold-treated at 4° C.in the dark for two days to promote the uniform germination of seeds.The plates were then transferred to 22° C. under continuous fluorescentlight illumination. After about 3 weeks, 16 kanamycin-resistantseedlings were transplanted into soil. These T₂ generation plants weregrown to maturity and individually harvested. Seed sterilization andscreening were repeated for these 16 plants. Only those T₃ generationseedlings that shown 95% and above survivor rate on the kanamycinselection plate were considered as homozygous line. These plants werethen transplanted into soil for seeds collection.

PCR Analysis of Transformant

Genomic DNA was extracted from Arabidopsis leaves carrying leaf-specificpromoter according to the method described by Doyle and Doyle (1990).PCR amplification was performed using primers specific for the GUS genewhich are GUS3FOR (5′TGACGCATGTCGCGCAAGAC3′; SEQ ID NO:27) and GUS2REV(5′ATCCTTTCGCACGTAAGTCC3′; SEQ ID NO:28). In this study, genomic DNAfrom wild type Arabidopsis was taken as negative control.

Histochemical GUS Assay of Transformant

GUS activity was measured histochemically following the method describedby Jefferson et al. (1987). The whole plant of Arabidopsis seedling (22days) carrying leaf-specific promoter were incubated infilter-sterilized GUS staining buffer (0.1 M sodium phosphate buffer,pH7.0; 1 mg/ml 5-bromo-4-chloro-3-indoyl-glucuronide/X-gluc and 0.2%Triton X-100) overnight at 37° C. in the dark. After staining,chlorophyll was removed from the plant using 70% ethanol. The presenceof blue precipitate were observed using NIKON SMZ800 Stereomicroscope.Blue deposits due to GUS activity in the transformant was compared withArabidopsis carrying 35S constitutive promoter and also wild type plant.

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While particular embodiments of the subject invention have beendescribed, it will be obvious to those skilled in the art that variouschanges and modifications to the subject invention can be made withoutdeparting from the spirit and scope of the invention. It is intended tocover, in the appended claims, all such modifications that are withinthe scope of this invention.

1. An isolated nucleic acid comprising the sequence of SEQ ID NO:
 1. 2.An isolated nucleic acid effective to regulate expression of an operablylinked polynucleotide, wherein the nucleic acid comprises a fragment ofthe nucleic acid of SEQ ID NO:1 with promoter activity, and wherein thefragment comprises the following elements depicted in FIG. 12: GATA Ibox, GATA II box, CCAAT box, and the G-box.
 3. A nucleic acid constructcomprising the isolated nucleic acid of claim 1 operably linked to arecombinant nucleic acid, wherein the nucleic acid construct whenintroduced into leaf tissues and/or photosynthetic tissues, stimulatesexpression of the gene recombinant nucleic acid in the leaf tissuesand/or photosynthetic tissues.
 4. A nucleic acid construct comprisingthe isolated nucleic acid of claim 2 operably linked to a recombinantnucleic acid, wherein the nucleic acid construct when introduced intoleaf tissues and/or photosynthetic tissues, stimulates expression of thegene recombinant nucleic acid in the leaf tissues and/or photosynthetictissues.
 5. The isolated nucleic acid of claim 2, wherein the fragmentof the nucleic acid of SEQ ID NO:1 further comprises one or morepromoter elements as depicted in FIG. 12 selected from the groupconsisting of: initiator element (Inr), I-box, wound responsive element(WUN), abscisic acid responsive element (ABA), and heat-shock responsiveelement (HSE).